The present invention relates to the homogeneous sulfation of polysaccharides and semisynthetic derivatives thereof, in particular glycosaminoglycans such as hyaluronic acid and its esters and tetraalkylammonium salts, for the preparation of new biomaterials useful in biomedical, health care, and pharmaceutical applications, and to such biomateriale per se. Such sulfated derivatives exhibit anti-thrombotic activity as evidenced by the lengthening of both the thrombin time and the whole blood clotting time. Moreover, the absence of hemolysis and the growth and shape of endothelial cells placed in contact with such sulfated derivatives indicate that these materials are promising heparin-like compounds.
Many molecules of biological origin are polyelectrolytes, and their interactions are very important in a wide variety of biochemical reactions. Consequently, synthetic and/or semisynthetic polyelectrolytes have been in use for some time now. These polyelectrolytes mimic the biological characteristics of natural polyelectrolytes, and can have somewhat different characteristics compared to the starting material.
Polyelectrolytes of biological origin include sulfated polysaccharides, and in particular, heparin and its derivatives (D. A. Lane and U. Lindahl, Eds., Heparin-Chemical and Biological Properties, Clinical Applications, Edward Arnold, London), which play an important role in cell-substrate interactions, particularly in the process of viral activity inhibition, in the process of blood coagulation, in lipid removal, etc.
Heparin is the most biologically reactive member of the family of sulfated glycosaminoglycans. It is well known for its antithrombotic and anticoagulant properties. In fact, it is extensively used in the management of cardiovascular diseases anti contributes enormously to the success of open heart surgery. Nevertheless, the structure of heparin is not simple and, due to the number of variations, is not entirely known. Commercial heparins consist of a spectrum of 21 heparins (Nader et al. (1974) Biochem. Biophys. Res. Commun. 57:488) ranging in molecular weights from 3,000 to 37,500 in varying anticoagulant activities.
The blood anticoagulant activity of heparin is attributed to structural features, e.g., degree of sulfation, degree of dissociation, particular sequences of COOxe2x88x92 and SOxe2x88x923 groups, as well as to molecular shape and size. These factors appear to be related to biological activity by virtue of their importance in the ion binding capacity of heparin (Stivala et al. (1967) Arch. Biochem. Biophys. 122:40). By virtue of its highly negatively charged nature, heparin has a strong affinity for cations, and its activity is pH-dependent.
Most of the readily available natural polysaccharides have been sulfated in an attempt to obtain heparin analogues (Hoffman et al. (982) Carbohydrate Res. 2:115; Kindness et al. (1980) Brit. J. Pharmac. 63:675; Horton et al. (1973) Carbohydrate Res. 30:349; Okada et al. (1979) Makromol. Chem. 180:813; Kikuchi et al. (1979) Nippon Kagaku Kaishi 1:127; Manzac et al. (1981) Proc. Third M.I.S.A.O. 5:504), and recently, sulfate, carboxylic, and sulfonate groups were attached to synthetic polymers such as polystyrene (Kanmaugue et al. (1985) Biomaterials 6:297) and polyurethane (Ito et al. (1992) Biomaterials 13:131). The anticoagulant activities of these materials were much lower than that heparin, and were dependent on the type and binding of the substituents, the degree of substitution, and sequences.
Some chemical reactions are known which make it possible to sulfate polysaccharides (WO 88/00211; EP 0 340 628; Nagasawa et al. (1986) Carbohydrate Research 158:183-190), but it has not yet been possible to obtain sulfated polysaccharides which, besides the chemical and chemical-physical characteristics peculiar to such polysaccharides, also possess new characteristics, such as anticoagulant activity.
The present approach to studying the structural properties associated with the anticoagulant properties of polysaccharides was first to choose polymers possessing well-defined chemical groups consisting of regular repeating units, and secondly to modify their chemical structure.
Such molecules must therefore:
(1) Contain regular sequences of monomeric units, and
(2) Be chemically modifiable without destroying their structure.
Hyaluronic acid, the major component of the mammalian extracellular matrix, consists of alternating units of N-acetylglucosamine and glucuronic acid residues, and therefore seems a suitable macromolecule.
The sulfation of alcoholic hydroxyls present in the polymeric chain of a polysaccharide or of one of its semisynthetic derivatives by the use of a suitable sulfating agent can lead to the formation of new derivatives with chemical-physical characteristics, but most of all biological characteristics, which are different from those of the starting material.
The polyelectrolyte polyssaccharides which can be used as substrates in the present invention include glycosaminoglycans. First and foremost among these is hyaluronic acid and the semisynthetic derivatives thereof. Some particularly important semisynthetic derivatives of hyaluronic acid are esters thereof with alcohols of the aliphatic, araliphatic, heterocyclic and cycloaliphatic series, designated xe2x80x9cHYAFF,xe2x80x9d that are described in U.S. Pat. Nos. 4,851,521, 4,965,353, and 5,202,431, and EP 0 216 453. Sulfation of such pre-processed biomaterials is a novel feature of the present invention. In this case, the sulfation reaction no longer occurs in the homogeneous phase, but rather on the surface of the biomaterial in the heterogeneous phase, activating the exposed hydroxyl groups toward the reaction solvent.
The degree of sulfation that can be obtained directly on the biomaterial is an important characteristic, and requires careful kinetic control. To avoid the solubilization of the biomaterial, induced by the increased hydrophilic nature of the polymer which constitutes the matrix, the number of xe2x80x94SO3 groups per dimeric unit must not exceed a certain level, generally less than 1.5-2, depending upon the degree of hydrophilicity of the starting biomaterial. For example, in the case of HYAFF 11 films, wherein all the carboxyls are involved in ester bonding with benzyl groups, the maximum degree of sulfation should not exceed 1.5.
The reagents commonly used for sulfation include the complex between sulfur trioxide and pyridine (SO3-pyridine).
The reaction is conducted by adding the sulfating reagent to a tetrabutylammonium salt of a polysaccharide in solution, or to a solution of a polysaccharide ester, which, in the case of partial esters, contains the remaining carboxy functions in the form of tetrabutylammonium salts, in aprotic solvents such as dimethylsulfoxide, N,Nxe2x80x2-dimethylformamide, and N-methylpyrrolidone in the temperature range of from about 0xc2x0 C. to about 60xc2x0 C.
Different degrees of Bulfation, measured by the number of sulfate groups per disaccharide unit, are obtained by varying the quantity of SO3-pyridine. The ratio between moles of hydroxyls and moles of sulfating reagent can vary between 1:1 and 1:12.
Surprisingly, the present inventors succeeded in sulfating the polysaccharide chain of hyiluronic acid and its semisynthetic derivatives in a specific and homogeneous manner without causing loss of the polymer""s characteristics, in particular its molecular weight, thus obtaining new polymers with biological and physico-chemical characteristics which hyaluronic acid and its semisynthetic derivatives did not previously possess.
By this method, it is possible to obtain new polymers with different levels of sulfation, but with the same molecular weight. Polymers with new biological characteristics can be obtained by using as starting materials biopolymers wherein the carboxy groups are salified with tetrabutylammonium salt. Such biopolymrs are not hemolytic.
A notable characteristic of these sulfated polysaccharides is their ability to increase blood coagulation time. The thrombin time test is performed by measuring how long it takes for fibrinogen to turn to fibrin once thrombin has been added to a sample of human blood in the presence of the test material. The thrombin time test in the same blood sample, but in the presence of the polymer used as starting material, is taken as a reference value. The test loses significance at over 240 seconds. The coagulation time is determined by simply measuring the time taken for a sample of human blood to coagulate in the presence of the test material. Times exceeding two hours are not considered.
Using the new biopolymers of the present invention, it is possible to develop new biomaterials for use in the biomedical, health-care, and pharmaceutical fields. The products obtained possess biocompatible and biological characteristics such as antithrombotic, anticoagulant, and antiviral activities. For example, sulfated polyanions have been shown to exhibit antiviral activity, including HIV inhibition. The new biopolymers of the present invention can also be used to advantage in cell growth processes, in controlled drug release systems, and more generally, in internal surgery, in extracorporeal oxygen circulation, in adhesion prevention, in permanent and biodegradable implants, and in dialysis.
For example, as in the case of other sulfated polymers, such as dextrans, sulfated hyaluronic acid having a molecular weight in the range of between about 10,000 and about 50,000 Daltons inhibits the production of tumor necrosis factor (TNF), which is the main target in the proliferation of inflammatory cells. Sulfated hyaluronic acid can therefore be used as a local anti-inflammatory agent in the form of hyaluroric acid-based biomaterials or compositions.
The new polymers can therefore be prepared in the form of gels, creams, or ointments, and can be used to produce biomaterials in the form of threads, sponges, gauzes, membranes, guide channels, non-woven fabrics and microspheres, according to the therapeutic uses for which they are intended. Lastly, depending upon the degree of sulfation and the molecular weight of the polymer, it is possible to produce polymers exhibiting antiviral activity and/or which can be use to intervene in the various stages of cell interactions. These biopolymers can also be used in coating processes, lending new biological properties to the surface of support material such as biomedical objects and devices.
Such sulfated biomaterials can be employed in applications where the product comes into contact with the blood or highly vascularized tissues, e.g., the use of biopolymeric dialysis tubes or membranes for internal or external surgery, which are capable of reducing cell adhesion, etc. In particular, the new, soluble sulfated hyaluronic acid derivatives of the present invention can be employed in the wide variety of applications already well known in the art for hyaluronic acid-based biomaterials.
For example, while hyaluronic acid derivatives having a degree of sulfation greater than 2.5 exhibit good anticoagulant activity, the molecular weight of the starting polymer can also be significant in influencing the properties of the new sulfated biopolymers of the present invention.
In particular, at least four sulfated hyaluronic acid derivatives are notable due to their molecular weight and degree of sulfation. These are:
1. Hyaluronic acid having a molecular weight in the range between about 10,000 and about 50,000 Daltons, and having a degree of sulfation of 2.5, 30, or 3.5;
2. Hyaluronic acid having a molecular weight in the range between about 50,000 and about 250,000 Daltons, and having a degree of sulfation of 2.5, 3.0, or 3.5;
3. Hyaluronic acid having a molecular weight in the range between about 250,000 and about 750,000 Daltons, and having a degree of sulfation of 2.5, 3.0, or 3.5; and
4. Hyaluronic acid having a molecular weight in the range between about 750,000 and about l,250,000 Daltons, and having a degree of sulfation of 2.5, 3.0, or 3.5.
The hyaluronic acid fractions having the molecular weights described above can be obtained by the use of membranes with particular molecular weight cut-off points, as is known in the art.
Among the semisynthetic ester derivatives of hyaluronic acid, polymeric matrices of HYAFF 11 (100% benzyl ester of hyaluronic acid) sulfated to degrees of 1.0 and 1.5, and HYAFF 11p75 (75% benzyl ester cf hyaluronic acid) sulfated to degrees of 0.5 and 1.0, are particularly interesting.
Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.